Method of differentiating hematopoietic stem cells into natural killer cells using YC-1 or IL-21

ABSTRACT

The present invention relates to an agent for differentiating hematopoietic stem cells into natural killer cells and a method for the differentiation, more precisely an agent for differentiating hematopoietic stem cells into natural killer cells comprising YC-I [3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] or IL-21 (Interleukin-21) as an active ingredient and a method for differentiating hematopoietic stem cells into natural killer cells using the same. The YC-I and IL-21 regulate the differentiation of hematopoietic stem cells into natural killer cells and increase the killing activity of NK cells. Therefore, the agent for NK cell differentiation of the present invention can be effectively used for cell therapy for the treatment of cancer by regulating the differentiation of hematopoietic stem cells into natural killer cells having tumor cell killing activity.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the §371 U.S. National Stage of International Application No.PCT/KR2007/004816, filed Oct. 2, 2007, which was published in Englishunder PCT Article 21(2), which in turn claims the benefit of KoreanPatent Application No. 10-2007-0077858, filed Aug. 2, 2007.

TECHNICAL FIELD

The present invention relates to an agent for differentiatinghematopoietic stem cells into natural killer cells and a method thereof,more precisely an agent for differentiating hematopoietic stem cellsinto natural killer cells comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] or IL-21(Interleukin-21) as an active ingredient and a method fordifferentiating hematopoietic stem cells into natural killer cells usingthe same.

BACKGROUND ART

Hematopoietic stem cells, kind of adult stem cells, have a potential fordifferentiating into almost every blood forming cells (includingerythrocytes, leucocytes, platelets and lymphocytes) and are constantlyauto-regenerated as immune cells from the hematopoietic stem cell inbone marrow. The cells forming immune system, in particular naturalkiller cells (referred as “NK cells” hereinafter), have an ability tokill tumor cells non-specifically.

This tumor cell killing activity of NK cells has been used for thetreatment of a solid tumor using lymphokine activated killer cells (LAK)and tumor infiltration lymphocytes (TIL) or for the immune therapy viadonor lymphocyte infusion (Itoh K, et al., J. Immunol., 136: 3910-3915,1986; Bordignon C, et al., Hematologia, 84: 1110-1149, 1999), which hasdrawn our attention as an advanced cell therapy for preventing rejectionfrom bone marrow transplantation or organ transplantation. It was alsoreported that the defect in NK cell differentiation or activity isrelated to various diseases including breast cancer (Konjevic G, et al.,Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma (Ryuke Y, etal., Melanoma Res., 13: 349-356, 2003) and lung cancer (Villegas F R, etal., Lung Cancer, 35: 23-28, 2002). So, correction of NK cell functionsseems to lead the way to treat these diseases.

NK cells are derived from hematopoietic stem cells in bone marrow. Thedifferentiation from hematopoietic stem cells into NK cells is composedof many steps, which have not been completely explained, yet.

IL-21 (Interleukin-21) is a cytokine secreted by activated CD4+ T cells(Warren J., et al., Nature, 5: 688-697, 2005). IL-21 receptor (IL-21R)is expressed in lymphocytes such as dendrite cells, NK, T, and B cells(Takaki R., et al., J. Immunol., 175: 2167-2173, 2005). The structure ofIL-21 is very similar to those of IL-2 and IL-15, and IL-21R sharesγ-chain with IL-2R, IL-15, IL-7R or IL-4R (Ensminger S M, et al., J.Immunol. 167 (1):532-541, 2001).

According to the previous reports, IL-21 induces maturation of NK cellprecursor in bone marrow (Parrish-Novak J., et al., Nature, 408: 57-63,2000) and characteristically increases effector functions of NK such ascytokine generation ability and killing activity (Strengell M, et al.,J. Immunol., 170: 5464-5469, 2003; Brady J, et al., J. Immunol., 172:2048-2058, 2004). IL-21 also increases effector functions of CD8+ Tcells, leading to the promotion of anticancer response of innate oradaptive immune system (Takaki R., et al., J. Immunol., 175: 2167-2173,2005; Moroz A., et al., J Immunol, 173: 900-909, 2004). In addition,IL-21 activates NK cells separated from human peripheral blood(Parrish-Novak J., et al., Nature, 408: 57-63, 2000) and plays animportant role in differentiation of NK cells from hematopoietic stemcells separated from umbilical cord blood (Sonia A. P, et al., Int.immunol., 18: 49-58, 2006).

YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] was firstdeveloped as an agent for treating circulatory diseases after it wasproved to inhibit coagulation of platelets and activate soluble GC(guanylyl cyclase) to inhibit contraction of blood vessel (Ko F N, etal., Blood 84. No. 12:4226-4233 1994; Teng C M, et al., Eur J Pharmacol,320:161-6, 1997; Galle J., et al., Br J Pharmacol, 127: 195-203, 1999).It was additionally found in recent studies that it inhibits HIF-1αaccumulation induced in hypoxia, reduces expressions of HIF-1 targetgenes (VEGF, erythropoietin, etc.) (Chun Y S, et al., Biochem Pharmacol,61:947-954, 2001), and inhibits tumor growth and angiogenesis in animals(Yeo E J, et al., J Natl Cancer Inst, 95: 516-525, 2003; Pan S L, etal., J pharmacol Exp Ther, 314:35-42, 2005). So, YC-1 becomes a leadingcompound for the development of an anticancer agent targeting HIF-1α(Yeo E J, et al., Cancer Res, 66: 6345-6352, 2006). Even though YC-1 isvery useful for the study of HIF-1α (Moeller B J, et al., Cancer cell 5:429-441, 2004; Funasaka T, et al., FASEB J, 19:1422-1430, 2005), themechanism of anticancer activity of YC-1 has not been disclosed, yet(Sun. H. L., et. al. Oncogene 1-11, 2006).

The present inventors completed this invention by confirming that IL-21(Interleukin-21) and YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] promote thedifferentiation of hematopoietic stem cells into natural killer cellsand increase the killing activity of natural killer cells.

DISCLOSURE Technical Problem

It is an object of the present invention to provide an agent fordifferentiating hematopoietic stem cells into natural killer cells and amethod for the differentiation.

Technical Solution

To achieve the above object, the present invention provides an agent fordifferentiating hematopoietic stem cells into natural killer cellscomprising YC-1 [3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] as anactive ingredient.

The present invention also provides an anticancer agent comprising IL-21(Interleukin-21) as an active ingredient.

The present invention further provides an agent for differentiatinghematopoietic stem cells into natural killer cells comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] and IL-21(Interleukin-21) as active ingredients.

The present invention also provides an anticancer agent comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] and IL-21(Interleukin-21) as active ingredients.

The present invention also provides a method for differentiatinghematopoietic stem cells into natural killer cells using IL-21 and/orYC-1.

The present invention also provides a cell therapy method for thetreatment of cancer, containing the step of administering natural killercells differentiated by the above method to tumor cells.

The present invention also provides a method for increasing the killingactivity of NK cells, containing the step of administering IL-21(Interleukin-21) to hematopoietic stem cells.

The present invention also provides a method for increasing the killingactivity of NK cells, containing the step of administering YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] to hematopoietic stemcells.

The present invention also provides a method for increasing the killingactivity of NK cells, containing the step of administering YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] and IL-21(Interleukin-21) to hematopoietic stem cells.

The present invention also provides a cell therapy method for thetreatment of cancer, containing the step of administering natural killercells with the improved tumor cell killing activity to tumor cells.

The present invention also provides a use of YC-1 for the preparation ofan agent for differentiating hematopoietic stem cells into naturalkiller cells or an anticancer agent.

The present invention also provides a use of IL-21 for the preparationof an anticancer agent.

In addition, the present invention provides a use of YC-1 and IL-21 forthe preparation of an agent for differentiating hematopoietic stem cellsinto natural killer cells or an anticancer agent.

Advantageous Effect

YC-1 and IL-21 are involved in NK cell differentiation and increase thekilling activity of NK cells. So, an agent for differentiatinghematopoietic stem cells into natural killer cells comprising YC-1 orIL-21 as an active ingredient and an anticancer agent comprising IL-21as an active ingredient can be effectively used for the development of anovel method for cell therapy for cancer and for the regulation of NKcell differentiation.

DESCRIPTION OF DRAWINGS

The application of the preferred embodiments of the present invention isbest understood with reference to the accompanying drawings, wherein:

FIG. 1 is a graph illustrating the purity of CD34⁺ hematopoietic stemcells measured by flow cytometry (the number written in quadrantindicates the purity).

FIG. 2 is a schematic diagram illustrating the process of thedifferentiation of hematopoietic stem cells separated from human cordblood into mature NK (mNK) cells through NK precursors (pNK):

-   -   HSC: hematopoietic stem cells;    -   SCF: stem cell factor; and    -   Flt3L: FMS-like tyrosine kinase 3 ligand.

FIG. 3 is a graph illustrating the expression of the surface molecule ofeach NK cell differentiation stage (the number written in quadrantindicates the purity).

FIG. 4 is a graph illustrating the result of FACS. In differentiation ofNK cells, IL-21 was treated to pNKs (NK precursor cells), which werecultured in the presence of IL-15 until they became differentiated intomature NK cells, followed by examination by FACS.

FIG. 5 is a graph illustrating the population of CD56+ NK cells overdifferentiation time, which is represented by %. Precisely, in NKdifferentiation, IL-21 was treated to pNKs, which were cultured in thepresence of IL-15 and CD56+ NK cells were counted over cultivation time.

FIG. 6 is a graph illustrating the result of ⁵¹Cr release assay. In NKdifferentiation, IL-21 was treated to pNKs, which were cultured in thepresence of IL-15 until they became differentiated into mature NK (mNK)cells, followed by ⁵¹Cr release assay:

-   -   E:T: effector cell:target cell.

FIG. 7 is a graph illustrating the result of FACS analysis. In NKdifferentiation, YC-1 was treated to pNKs, which were cultured in thepresence of IL-15 until they became differentiated into mature NK (mNK)cells, followed by FACS.

FIG. 8 is a graph illustrating the population of CD56+ NK cells.Precisely, in NK differentiation, YC-1 was treated to pNKs, which werecultured in the'presence of IL-15. CD56+ NK cells were counted over thecultivation time and represented by %.

FIG. 9 is a graph illustrating the result of ⁵¹Cr release assay. In NKdifferentiation, YC-1 was treated to pNKs, which were cultured in thepresence of IL-15 until they became differentiated into mature NK (mNK)cells, followed by ⁵¹Cr release assay:

-   -   E:T: effector cell:target cell.

FIG. 10 is a graph illustrating the result of FACS analysis. In NKdifferentiation, both IL-21 and YC-1 were treated to pNKs, which werecultured in the presence of IL-15 until they became differentiated intomature NK (mNK) cells, followed by FACS.

FIG. 11 is a graph illustrating the population of CD56+ NK cells overcultivation time. In NK differentiation, both IL-21 and YC-1 weretreated to pNKs, which were cultured in the presence of IL-15, followedby measuring the population of CD56+ NK cells over cultivation time.

MODE FOR INVENTION

Hereinafter, the present invention is described in detail.

The present invention provides an agent for differentiatinghematopoietic stem cells into natural killer cells comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] as an activeingredient.

The YC-1 (C₁₉H₁₆N₂O₂) is represented by formula 1.

The present invention also provides an anticancer agent comprising IL-21(Interleukin-21) as an active ingredient.

The IL-21 (Interleukin-21) is preferably selected from the groupconsisting of (a) the protein comprising the amino acid sequencerepresented by SEQ. ID. NO: 1; (b) the protein encoded by the DNAcontaining the coding region of the nucleotide sequence represented bySEQ. ID. NO: 2; (c) the protein comprising the amino acid sequence withsubstitution, deletion, insertion and/or addition of one or more aminoacids in the amino acid sequence represented by SEQ. ID. NO: 1 and isfunctionally equal to the protein comprising the amino acid sequencerepresented by SEQ. ID. NO: 1; and (d) the protein encoded by the DNAhybridized with the DNA comprising the nucleotide sequence representedby SEQ. ID. NO: 2 under the strict condition and is functionally equalto the protein comprising the amino acid sequence represented by SEQ.ID. NO: 1.

Hybridization under the strict condition enables the selection of DNAhaving the nucleotide sequence with high homology. Thus, the chances arehigh for the separated protein therefrom to be a protein that isfunctionally equal to IL-21. The nucleotide sequence with high homologymeans, for example, the nucleotide sequence having at least 70% homologywith the nucleotide sequence represented by SEQ. ID. NO: 2, preferablyhaving at least 80% homology and more preferably at least 90% and mostpreferably at least 95% homology with the nucleotide sequencerepresented by SEQ. ID. NO: 2. In the case of amino acid sequence, theamino acid sequence having at least 70% homology, preferably at least80% homology, more preferably at least 90% and most preferably at least95% homology with the amino acid sequence represented by SEQ. ID. NO: 1can be selected. The percent of homology can be determined by theconventional algorithm selected by those in the art.

The hybridization can be performed by DNA-DNA hybridization under thestrict condition (Hames and Higgins, Eds. (1985) Nucleic AcidHybridization, IRL Press, U.K.) known to those in the art and thecondition can be determined in the washing process after hybridization.

The present invention further provides an agent for differentiatinghematopoietic stem cells into natural killer cells comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] and IL-21(Interleukin-21) as active ingredients.

The present inventors separated CD34+ hematopoietic stem cells (referredas “HSC cells” hereinafter) with 92% purity from cord blood (see FIG.1). The HSC cells were differentiated into mature NK cells through pNKs(see FIG. 2) and the expression of the surface molecule was examined foreach NK cell differentiation stage (see FIG. 3). As a result, CD56 cellswere increased.

In NK cell differentiation, IL-21 was treated to pNKs, which werecultured in the presence of IL-15 to differentiate pNKs into mNK cells.FACS analysis was performed. As a result, CD56+ NK cell population wasincreased in IL-21 treated group, compared with the IL-21 non-treatedcontrol (treated with IL-15 alone) (see FIGS. 4 and 5), suggesting thatIL-21 was involved in NK differentiation. From the result of ⁵¹Crrelease assay, it was confirmed that IL-21 treated group exhibitedincreased killing activity, compared with IL-21 non-treated group(treated with IL-15 alone) (see FIG. 6), suggesting that IL-21 wasinvolved in the activation of the killing activity of NK cells.

NK precursor cells (pNK) were treated with YC-1 and cultured in thepresence of IL-15 to differentiate into matured NK (mNK) cells, followedby FACS analysis. As a result, CD56+ NK cells were increased in YC-1treated group, compared with YC-1 non-treated group (treated with IL-15alone) (see FIGS. 7 and 8), suggesting that YC-1 was involved in NK celldifferentiation. From the result of ⁵¹Cr release assay, it was confirmedthat YC-1 treated group exhibited increased killing activity, comparedwith IL-21 non-treated group (treated with IL-15 alone) (see FIG. 9),suggesting that YC-1 was involved in the activation of the killingactivity of NK cells.

NK precursor cells (pNK) were treated with both IL-21 and YC-1 at thesame time and NK cell differentiation was investigated. As a result, NKcell differentiation was significantly increased (see FIGS. 10 and 11).

As explained hereinbefore, IL-21 and YC-1 promote differentiation fromhematopoietic stem cells into natural killer cells and increase thekilling activity of NK cells, so that they can be effectively used asagents for differentiating hematopoietic stem cells into NK cells.

The present invention also provides an anticancer agent comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] and IL-21(Interleukin-21) as active ingredients.

The agent for differentiating into NK cells and the anticancer agent ofthe invention can be used for cell therapy for the treatment of cancer.

The cancer herein is preferably selected from the group consisting ofbreast cancer, melanoma, stomach cancer, liver cancer, blood cancer,colon cancer, and lung cancer, but not always limited thereto.

If there is a defect in the differentiation and activity of NK cells,various cancers might be developed, for example, breast cancer (KonjevicG, et al., Breast Cancer Res. Treat., 66: 255-263, 2001), melanoma(Ryuke Y, et al., Melanoma Res., 2003, 13: 349-356) and lung cancer(Villegas F R, et al., Lung Cancer, 35: 23-28, 2002), etc, according tothe previous reports. Therefore, it is expected to treat such cancers asthe above by regulating NK cell differentiation using the agent for NKcell differentiation and the anticancer agent of the invention.

The agent for NK cell differentiation and the anticancer agent of thepresent invention can be administered orally or parenterally and be usedin general forms of pharmaceutical formulation. The agent for NK celldifferentiation and the anticancer agent of the present invention can beprepared for oral or parenteral administration by mixing with generallyused fillers, extenders, binders, wetting agents, disintegrating agents,diluents such as surfactant, or excipients. Solid formulations for oraladministration are tablets, pills, dusting powders, granules andcapsules. These solid formulations are prepared by mixing one or moresuitable excipients such as starch, calcium carbonate, sucrose, lactose,gelatin, etc. Except for the simple excipients, lubricants, for examplemagnesium stearate, talc, etc, can be used. Liquid formulations for oraladministrations are suspensions, solutions, emulsions and syrups, andthe above mentioned formulations can contain various excipients such aswetting agents, sweeteners, aromatics and preservatives in addition togenerally used simple diluents such as water and liquid paraffin.Formulations for parenteral administration are sterilized aqueoussolutions, water-insoluble excipients, suspensions, emulsions, andsuppositories. Water insoluble excipients and suspensions can contain,in addition to the active compound or compounds, propylene glycol,polyethylene glycol, vegetable oil like olive oil, injectable ester likeethylolate, etc. Suppositories can contain, in addition to the activecompound or compounds, witepsol, macrogol, tween 61, cacao butter,laurin butter, glycerol, gelatin, etc.

The effective dosage of the agent for NK cell differentiation or theanticancer agent of the present invention is 0.1˜0.2 mg/kg, and morepreferably 0.15 mg/kg, and the administration times are preferably 1˜3times a day.

The present invention also provides a method for differentiatinghematopoietic stem cells into natural killer cells using IL-21 and/orYC-1.

Particularly, the method for differentiating hematopoietic stem cellsinto NK cells comprises the following steps:

1) inducing proliferation of NK precursor cells by adding a NK precursorinducer to hematopoietic stem cells; and

2) differentiating the NK precursor cells into mature NK cells by addingIL-21 and/or YC-1 to the NK precursor cells of step 1).

In step 1), the “NK precursor inducer” means any substance that is ableto induce differentiation from hematopoietic stem cells into NKprecursor cells, which is preferably SCF or Flt3L, but not alwayslimited thereto.

In step 2), the preferable treatment amount of IL-21 is 10 ng/ml˜50ng/ml, and the treatment amount of YC-1 is 0.5 uM˜5 uM. In the case ofco-treatment of YC-1 and IL-21, preferable contents of YC-1 and IL-21are respectively 0.5 uM and 10 ng/ml, but not always limited thereto.

In step 2), the NK precursor cells are preferably cultured with IL-15(Interleukin-15), but not always limited thereto.

According to the conventional method, hematopoietic stem cells separatedfrom cord blood are treated with IL-15 and IL-21 simultaneously toinduce proliferation and differentiation at the same time. So,differentiation speed is very slow and thus takes long time (30 days).However, in the method of the present invention, differentiation fromhematopoietic stem cells into NK precursor cells is first induced byadding SCF and Flt3L and then differentiation into mature NK cells isinduced by adding IL-15 and IL-21 and/or YC-1, which makes the methodefficient and reduces differentiation time (14 days).

The present invention also provides a cell therapy method for thetreatment of cancer, containing the step of administering natural killercells differentiated by the above method to tumor cells.

The differentiation from hematopoietic stem cells into NK cells above isinduced in vitro.

The cancer herein is preferably selected from the group consisting ofbreast cancer, melanoma, stomach cancer, liver cancer, blood cancer,colon cancer, and lung cancer, but not always limited thereto.

To induce differentiation from hematopoietic stem cells into NK cells,YC-1 or IL-21 can be administered but co-administration of YC-1 andIL-21 is preferred.

The present invention also provides a method for increasing the killingactivity of NK cells, containing the step of administering IL-21(Interleukin-21) to hematopoietic stem cells.

The effective dosage of IL-21 is 10 ng/ml˜50 ng/ml, and is preferablyadministered together with IL-15 (Interleukin-15) for the culture, butnot always limited thereto.

The present invention also provides a method for increasing the killingactivity of NK cells, containing the step of administering YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] to hematopoietic stemcells.

The preferable dosage of YC-1 is 0.5 uM˜5 uM, and is preferablyadministered together with IL-15 (Interleukin-15) for the culture, butnot always limited thereto.

The present invention also provides a method for increasing the killingactivity of NK cells, containing the step of administering YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] and IL-21(Interleukin-21) to hematopoietic stem cells.

The preferable hematopoietic stem cells for the invention are those whoare under-going differentiation into NK cells.

The preferable doses of YC-1 and IL-21 are respectively 0.5 uM and 10ng/ml, which are preferably co-treated with IL-15 (Interleukin-15) forthe culture of hematopoietic stem cells, but not always limited thereto.

The present invention also provides a cell therapy method for thetreatment of cancer, containing the step of administering natural killercells with the improved tumor cell killing activity to tumor cells.

The killing activity of NK cells is increased in vitro.

The cancer herein is preferably selected from the group consisting ofbreast cancer, melanoma, stomach cancer, liver cancer, blood cancer,colon cancer, and lung cancer, but not always limited thereto.

To increase the killing activity of NK cells, YC-1 or IL-21 can betreated but co-administration of YC-1 and IL-21 is more preferred.

In addition, the present invention provides a use of YC-1 or IL-21 forthe preparation of an agent for differentiating hematopoietic stem cellsinto natural killer cells or an anticancer agent.

Practical and presently preferred embodiments of the present inventionare illustrative as shown in the following Examples.

However, it will be appreciated that those skilled in the art, onconsideration of this disclosure, may make modifications andimprovements within the spirit and scope of the present invention.

EXAMPLE 1 Separation of Hematopoietic Stem Cells from Cord Blood

Cord blood provided from Department of Obstetric & Gynecology, KonyangUniversity Hospital, Taejon, Korea, for the purpose of study only wasdiluted in RPMI 1640 (GIBCO-BRL, USA) at the ratio of 2:1. The preparedcord blood was loaded on the upper part of Ficoll-Paque (Sigma, USA),followed by centrifugation (20,000 rpm, 30 minutes) to separatemononuclear cells (MNC). Erythrocytes were eliminated from the obtainedcells and the obtained mononuclear cells were marked with thehematopoietic stem cell marker ‘CD34 microbeads’. Then, CD34+ cells wereseparated by MS/RS column and MACS (Magnetic Activated Cell Sorter). Thepurity of the obtained CD34+ hematopoietic stem cells (referred as “HSCcells” hereinafter) was measured by FACS (BD Bioscience, Mountainview,Calif.), which was 92% (FIG. 1).

EXAMPLE 2 Differentiation from Hematopoietic Stem Cells of Cord Bloodinto NK Cells

The HSC cells separated from cord blood in Example 1 were inoculated ina 12-well plate (Falcon, USA) containing the Myelocult complete medium(Stem cell Technology, CA) supplemented with 30 ng/ml of human Factor(PeproTech, USA), 50 ng/ml of human Flt3L (FMS-like tyrosine kinase 3ligand, PeproTech, USA), 5 ng/ml of human IL-7 (PeproTech, USA), and10⁻⁶ M of hydro SCF (Stem Cell hydrocortisone, Stem cell Technology, CA)at the concentration of 1×10⁶ cells/well, followed by culture for 14days in a 37° C., 5% CO₂ incubator. Three days later, half of thesupernatant was replaced with a fresh medium containing cytokines havingthe same composition as the above. For the differentiation into matureNK cells (referred as “NK cells” hereinafter), HSC cells were recovered14 days later and cultured again for 14 days in the presence of humanIL-15 (30 ng/ml, PeproTech, USA). Three days later, half of the mediumwas replaced with a fresh medium containing cytokines having the samecomposition as the above. (FIG. 2). On the 28^(th) day of culture, thepurity of NK cells was measured by using anti-CD56 antibody and theexpression of NK cell receptor was measured by flow cytometry (FACS)(FIG. 3).

EXAMPLE 3 Effect on NK Cell Differentiation of IL-21

To investigate the effect on NK cell differentiation of IL-21 known topromote differentiation and increase activity, IL-21 (20 ng/ml,PeproTech, USA) was treated during the process of differentiation fromhematopoietic stem cells of cord blood into mature NK cells (mNK)through NK precursor cells (pNK). The cells were cultured in thepresence of IL-15, followed by FACS analysis and ⁵¹Cr release assay.

IL-21 has the amino acid sequence represented by SEQ. ID. NO: 1 and thenucleotide sequence represented by SEQ. ID. NO: 2.

From the result of FACS analysis, it was confirmed that CD56+ NK cellpopulation was increased in IL-21 treated group (FIGS. 4 and 5).

From the result of ⁵¹Cr release assay, it was confirmed that the killingactivity of NK cells was increased by IL-21 (FIG. 6). E:T (effectorcell:target cell) was 2.5:1.

The above results indicate that IL-21 is involved in NK celldifferentiation and directly affects the killing activity of NK cells.

EXAMPLE 4 Effect on NK Cell Differentiation of YC-1

To investigate the effect on NK cell differentiation of YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole, formula 1] known as ananticancer agent functioning in suppressing HIF-1α activity to inhibittumor cell growth, YC-1 (1 uM, Sigma) was treated during the process ofdifferentiation from hematopoietic stem cells of cord blood into matureNK cells (mNK) through NK precursor cells (pNK). The cells were culturedin the presence of IL-15, followed by FACS analysis and ⁵¹Cr releaseassay.

From the result of FACS analysis, it was confirmed that CD56+ NK cellpopulation was increased in YC-1 treated group (FIGS. 7 and 8).

From the result of ⁵¹Cr release assay, it was confirmed that the killingactivity of NK cells was increased by YC-1 (FIG. 9). E:T (effectorcell:target cell) was 5:1.

The above results indicate that YC-1 is involved in NK celldifferentiation and directly affects the killing activity of NK cells.

EXAMPLE 5 Synergy Effect of IL-21 and YC-1 on NK Cell Differentiation

To investigate synergy effect of IL-21 and YC-1 on NK celldifferentiation, IL-21 (10 ng/ml) and YC-1 (0.5 uM) were co-treatedduring the process of differentiation from hematopoietic stem cells ofcord blood into mature NK cells (mNK) through NK precursor cells (pNK).The cells were cultured in the presence of IL-15, followed by FACSanalysis. At that time, the concentrations of IL-21 and YC-1 wereregulated low in order not to affect NK cell differentiation (10 ng/mlof IL-21 and 0.5 uM of YC-1).

From the result of FACS analysis, NK cells were slightly increased wheneach of the two was separately treated but NK cells were significantlyincreased when both of them were treated together (FIGS. 10 and 11),indicating that the co-treatment of IL-21 and YC-1 brings synergyeffect.

INDUSTRIAL APPLICABILITY

As explained hereinbefore, YC-1 and IL-21 regulate differentiation ofhematopoietic stem cells into natural killer cells and increase thekilling activity of NK cells. Therefore, an agent for differentiatinghematopoietic stem cells into natural killer cells comprising YC-1[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] or IL-21(Interleukin-21) as an active ingredient and a method fordifferentiating hematopoietic stem cells into natural killer cells usingthe same can be effectively used for cell therapy for the treatment ofcancer.

Those skilled in the art will appreciate that the conceptions andspecific embodiments disclosed in the foregoing description may bereadily utilized as a basis for modifying or designing other embodimentsfor carrying out the same purposes of the present invention. Thoseskilled in the art will also appreciate that such equivalent embodimentsdo not depart from the spirit and scope of the invention as set forth inthe appended claims.

1. A method for differentiating hematopoietic stem cells into naturalkiller (NK) cells comprising the following steps: (i) inducingproliferation of NK precursor cells by adding a NK precursor inducer tohematopoietic stem cells in vitro; and (ii) differentiating the NKprecursor cells into mature NK cells by adding:[3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole] (YC-1) andinterleukin-15 (IL-15); or YC-1, interleukin-21 (IL-21), and IL-15 tothe NK precursor cells.
 2. The method according to claim 1, wherein theNK precursor inducer of step (i) is stem cell factor (SCF) and FMS-liketyrosine kinase 3 ligand (Flt3L).
 3. The method according to claim 1,wherein the IL-21 of step (ii) is a protein selected from the groupconsisting of: (a) the protein comprising the amino acid sequencerepresented by SEQ ID NO: 1; and (b) the protein encoded by the DNAcontaining the coding region of the nucleotide sequence represented bySEQ ID NO:
 2. 4. The method according to claim 1, wherein the dosage ofthe IL-21 of step (ii) is 10 ng/ml-50 ng/ml.
 5. The method according toclaim 1, wherein the dosage of the YC-1 of step (ii) is 0.5 μM-5 μM.